Individual Dye Molecules Research Articles

Single-molecule tracking of dye diffusion in synthetic polymers: A tutorial review

Single-molecule tracking (SMT) methods have been widely employed to offer a high-resolution characterization of synthetic polymers under ambient conditions and, thus, have advanced our understanding of their unique material properties. SMT is based on the systematic monitoring of the diffusive motions of individual fluorescent dye molecules in the as-prepared polymer thin films or thicker monoliths. Quantitative assessment of the recorded SMT video data involves the systematic analysis of the generated diffusion trajectories of a single molecule using well-established and reported methods. The results have offered a wealth of new information on the structural alignment, orientational order, and long-range continuity of the polymer microdomains; the nanoscale material heterogeneities governed by defects, misalignment, and ill-controlled preparation conditions; as well as the various forms of probe–host interactions on the single-molecule level. In the first part of this Tutorial review, we describe the fundamental principles and instrumentation of SMT, before offering interested readers and potential future SMT users a practical guidance on the selection of fluorescent probe molecules, preparation of suitable samples, and optimization of experimental conditions and imaging parameters. Then, we highlight several representative SMT studies in microphase-separated block copolymers, and semicrystalline and amorphous homopolymers to further emphasize the usefulness of SMT methods for polymer characterization without neglecting some of their shortcomings. This Tutorial review is written with the motivation to attract new researchers to the fast-growing field and assist them in starting their own SMT research of synthetic polymers and other technologically useful soft matter systems.

UV-selective organic absorbers for the cosensitization of greenhouse-integrated dye-sensitized solar cells: synthesis and computational study.

Molecular cosensitization is favorable for manipulating solar radiation through the judicious choice of cosensitizers having complementary absorption spectra. For greenhouse-integrated dye-sensitized solar cells (DSCs), the manipulation of solar radiation is crucial in order to maximize the flow of photosynthetically active radiation (PAR) for the effectual photosynthetic activity of plants; meanwhile, non-PAR is utilized in agrivoltaics for generating electricity. In this study, we report the synthesis of novel four UV-selective absorbers, based on the diimide scaffold, functionalized with carboxylate and pyridyl anchoring groups, for adequate adsorption onto the TiO2 electrode in DSC. The UV/Vis absorption spectra of the DMF solution-based free dyes were measured experimentally. Basic photophysical and energetics requirements for operating greenhouse-integrated DSCs were examined at the molecular level via (time-dependent) density functional theory-based calculations. The computational results revealed the outperformance of the biphenyldiimide-structured DI-CA1 dye, especially for maximum charge transferred to its anchor, lower thermodynamic barrier for dissociating the photogenerated exciton, largest Stokes' shift, strong electronic coupling with TiO2 nanoparticles, and higher degree of charge separation at the DI-CA1/TiO2 interface. PDOS showed deeper existence for the LUMO level in the CB of TiO2, which expedites the electron injection process. The chemical and optical compatibility of DI-CA1 were then investigated as a potential cosensitizer of a reference BTD–DTP1, a green light-absorbing dye. Considerable overlap between the fluorescence spectrum of DI-CA1 and absorption spectrum of the reference BTD–DTP1 advocated the opportunity of excitation energy transfer via the radiative trivial reabsorption mechanism, which confirms the cosensitization functionality. Energy decomposition analysis and reduced density gradient maps estimated the chemical compatibility owing to weak dispersion interactions as the dominant stabilizing attractive force. This noncovalent functionalization retains the chemical compatibility without distorting the π–π conjugation and the associated physicochemical properties of the individual dye molecules. Along with the expanded consumption of non-photosynthetically active solar radiation, an improved power conversion efficiency of greenhouse-integrated DSC is accordingly expected.

Air–Water Interface Assembly of Protein Nanofibrils Promoted by Hydrophobic Additives

Herein we demonstrate a novel way of modifying the colloidal stability of proteins by the presence of hydrophobic molecules. A protein capable of self-assembly into protein nanofibrils (PNFs) is milled with a hydrophobic molecular material. Upon dissolution in acidic water followed by heating, the proteins are converted into PNFs containing hydrophobic dyes. When aqueous dispersions of such PNFs are heated, films are formed at the air–water interface. The films contain ordered, optically anisotropic domains, and the shape of the reaction vessel can influence the PNF packing. We demonstrate the generality of the process by employing PNFs derived from the three proteins bovine insulin (INS), β-lactoglobulin (BLG), and hen egg white lysozyme (HEWL) in combination with the dyes α-sexithiophene (6T) and 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM). As individual dye molecules are oriented along the long PNF axis, whole ensembles of dyes become aligned and, as a result, display emission of polarized light. Moreover, in the case of PNFs stained with DCM, stimulated emission is demonstrated.

A workflow for sizing oligomeric biomolecules based on cryo single molecule localization microscopy.

Single molecule localization microscopy (SMLM) has enormous potential for resolving subcellular structures below the diffraction limit of light microscopy: Localization precision in the low digit nanometer regime has been shown to be achievable. In order to record localization microscopy data, however, sample fixation is inevitable to prevent molecular motion during the rather long recording times of minutes up to hours. Eventually, it turns out that preservation of the sample's ultrastructure during fixation becomes the limiting factor. We propose here a workflow for data analysis, which is based on SMLM performed at cryogenic temperatures. Since molecular dipoles of the fluorophores are fixed at low temperatures, such an approach offers the possibility to use the orientation of the dipole as an additional information for image analysis. In particular, assignment of localizations to individual dye molecules becomes possible with high reliability. We quantitatively characterized the new approach based on the analysis of simulated oligomeric structures. Side lengths can be determined with a relative error of less than 1% for tetramers with a nominal side length of 5 nm, even if the assumed localization precision for single molecules is more than 2 nm.

Effect of metallic substrates and cavities on emission kinetics of dye-doped polymeric films

We have studied emission kinetics in dye-doped polymeric films (HITC:PMMA), deposited on top of glass and silver and embedded in Fabry–Perot cavities (metal-insulator-metal waveguides). For highly doped films on glass, we observed strong concentration quenching, as evidenced by a dramatic shortening of the emission kinetics, consistent with our previous studies. However, for the same dye-doped films on top of silver, slower emission kinetics were observed despite the high decay rates of individual dye molecules near the metallic surface. The concentration quenching rates in Fabry–Perot cavities were nearly identical to those of HITC:PMMA films deposited on top of silver. These findings are explained within a theoretical model for the inhibition of Förster energy transfer near a metallic surface. Furthermore, the emission kinetics of the dye-doped films on top of silver were approximately single exponential—consistent with the strong coupling of excited molecules with propagating surface plasmons.

Exciton Delocalization in Indolenine Squaraine Aggregates Templated by DNA Holliday Junction Scaffolds.

Exciton delocalization plays a prominent role in the photophysics of molecular aggregates, ultimately governing their particular function or application. Deoxyribonucleic acid (DNA) is a compelling scaffold in which to template molecular aggregates and promote exciton delocalization. As individual dye molecules are the basis of exciton delocalization in molecular aggregates, their judicious selection is important. Motivated by their excellent photostability and spectral properties, here, we examine the ability of squaraine dyes to undergo exciton delocalization when aggregated via a DNA Holliday junction (HJ) template. A commercially available indolenine squaraine dye was chosen for the study given its strong structural resemblance to Cy5, a commercially available cyanine dye previously shown to undergo exciton delocalization in DNA HJs. Three types of DNA-dye aggregate configurations-transverse dimer, adjacent dimer, and tetramer-were investigated. Signatures of exciton delocalization were observed in all squaraine-DNA aggregates. Specifically, strong blue shift and Davydov splitting were observed in steady-state absorption spectroscopy and exciton-induced features were evident in circular dichroism (CD) spectroscopy. Strongly suppressed fluorescence emission provided additional, indirect evidence for exciton delocalization in the DNA-templated squaraine dye aggregates. To quantitatively evaluate and directly compare the excitonic Coulombic coupling responsible for exciton delocalization, the strength of excitonic hopping interactions between the dyes was obtained by simultaneously fitting the experimental steady-state absorption and CD spectra via a Holstein-like Hamiltonian, in which, following the theoretical approach of Kühn, Renger, and May, the dominant vibrational mode is explicitly considered. The excitonic hopping strength within indolenine squaraines was found to be comparable to that of the analogous Cy5 DNA-templated aggregate. The squaraine aggregates adopted primarily an H-type (dyes oriented parallel to each other) spatial arrangement. Extracted geometric details of the dye mutual orientation in the aggregates enabled a close comparison of aggregate configurations and the elucidation of the influence of dye angular relationship on excitonic hopping interactions in squaraine aggregates. These results encourage the application of squaraine-based aggregates in next-generation systems driven by molecular excitons.

Theoretical investigation of auxiliary electronic acceptors in modifying D-D-π-A sensitizers for dye-sensitized solar cells.

In light of the performance of the SD2 pigments in DSSC, in order to expand the absorption spectral scope, decrease the energy difference between the highest occupied and the lowest unoccupied molecular orbitals, with SD2 dye molecular electron donor and electron acceptor as the fundamental framework, the indole fragment and thiophene derivative in the prototype dye molecule were replaced by the two π-bridges (labeled PA, PB, respectively) and the four auxiliary electron acceptors (labeled A1, A2, A3, A4, respectively). For the sake of characterizing dye molecules as thoroughly as possible in DSSC, the frontier orbital energy levels, ultraviolet absorption spectra, natural bond orbital analysis, intramolecular charge transfer, charge and hole reorganization energies, parameters influencing the short-circuit current density and the open-circuit photovoltage for these eight individual dye molecules are carried out to try to fully characterize the properties of these dye molecules. According to these computational results of physical quantities and based on the performance of these dye molecules in the above aspects, in this paper, six free molecular models were picked out to combine with titanium dioxide cluster to calculate their geometrical structures, frontier orbital distributions, electron excitation energies, ultraviolet absorption spectra and the composition of the electronic transitions in chloroform solvent with polarizable continuum model. The results of these calculations show that the PA-A2 and PB-A4 dye molecule has better properties in electron transfer and spectral absorption range before and after the adsorption on the titanium dioxide.

Tunable Plexcitonic Nanoparticles: A Model System for Studying Plasmon–Exciton Interaction from the Weak to the Ultrastrong Coupling Regime

Controlling the number of dye molecules on metallic nanoparticles, which in turn affects the magnitude of Rabi splitting energy, is crucial for obtaining hybrid metal core–organic shell nanoparticles with tunable optical properties in the visible spectrum since the magnitude of the Rabi splitting energy directly determines the strength of the coupling between plasmonic nanoparticles and dye molecules. In this work, we present a new method for the synthesis of plexcitonic nanoparticles, and thus we are able to control the number of dye molecules self-assembled on Ag nanoprisms (Ag NPs) by adjusting the concentration of dye molecules used in the synthesis. Indeed, individual dye molecules self-assemble into J-aggregates on Ag NPs. Thus, in the finite-element simulations and experimental data of the hybrid metal organic nanoparticles, we observed a transition from weak coupling to the ultrastrong coupling regime. Besides, ultrafast energy transfer between plasmonic nanoparticles and excitonic aggregated dye ...

Monitoring Nanoscale Deformations in a Drawn Polymer Melt with Single-Molecule Fluorescence Polarization Microscopy.

Elongating a polymer melt causes polymer segments to align and polymer coils to deform along the drawing direction. Despite the importance of this molecular response for understanding the viscoelastic properties and relaxation behavior of polymeric materials, studies on the single-molecule level are rare and were not performed in real time. Here we use single-molecule fluorescence polarization microscopy for monitoring the position and orientation of single fluorescent perylene diimide molecules embedded in a free-standing thin film of a polymethyl acrylate (PMA) melt with a time resolution of 500 ms during the film drawing and the subsequent stress relaxation period. The orientation distribution of the perylene diimide molecules is quantitatively described with a model of rod-like objects embedded in a uniaxially elongated matrix. The orientation of the fluorescent probe molecules is directly coupled to the local deformation of the PMA melt, which we derive from the distances between individual dye molecules. In turn, the fluorescence polarization monitors the shape deformation of the polymer coils on a length scale of 5 nm. During stress relaxation, the coil shape relaxes four times more slowly than the mechanical stress. This shows that stress relaxation involves processes on length scales smaller than a polymer coil. Our work demonstrates how optical spectroscopy and microscopy can be used to study the coupling of individual fluorescent probe molecules to their embedding polymeric matrix and to an external mechanical stimulus on the single-molecule level.

Sensitization of ZnO by Light-Harvesting Antennas Composed of Multiple Stacked Dyes Grown by Liquid-Phase Molecular Layer Deposition

Light-harvesting antennas in the photosynthesis systems of plants are known to be high-efficiency photon collectors. Here we use light-harvesting antennas in dye-sensitized solar cells.1 The concept of spectral sensitization of ZnO by light-harvesting antennas composed of multiple stacked dyes is shown in Fig. 1. Dyes with different energy gaps are stacked in a designated sequence so that a wide range of wavelengths are absorbed. The resulting broad absorption spectrum is a superposition of the narrow absorption bands of the individual dye molecules. This energy scheme is expected to decrease the heat generation caused by excess photon energy in the light absorption process.2 We have previously sensitized ZnO using two-dye stacked structures consisting of a p-type dye and an n-type dye to widen the photocurrent spectral width.2 In the present work, we investigate sensitization of ZnO using three-dye stacked light-harvesting antennas grown by liquid-phase molecular layer deposition (LP-MLD)2-4using n- and p-type dye molecules. Meier5reported that p-type dyes such as rose bengal (RB), eosin (EO) and fluorescein (FL) tend to have negative charge because they accept electrons, while n-type dyes like crystal violet (CV) and brilliant green (BG) typically have positive charge as they donate electrons. Because ZnO is an n-type material, as shown in Fig. 2, three-dye stacked structures can be grown using a single dye in each step by providing source molecules with a sequence of p-, n-, and p-type. Here, the LP-MLD process was achieved by electrostatic force. Namely, the attraction between p–n and repulsion between p–p and n–n molecules resulted in self-limiting growth, allowing us to construct structures of [n-type ZnO/p-type dye/n-type dye/p-type dye]. Samples for photocurrent measurements were prepared as follows. In step 1, after a ZnO powder layer (~5 μm thick) was formed on a glass substrate with a slit-type indium tin oxide (ITO) electrode (slit width of 60 μm), the layer was immersed in an isopropyl alcohol (IPA) solution of the first p-type dye (p1). In step 2, the layer was rinsed with IPA, and immersed in an IPA solution of n-type dye (n1) before the layer was dried. In step 3, the layer was immersed in an IPA solution of the second p-type dye (p2) in the same manner. This procedure gave three-dye stacked light-harvesting antennas with a structure of [ZnO/p1/n1/p2]. Photocurrent spectra were then measured by applying a 3-V potential across the slit-type ITO electrodes over a wavelength range of 400–700 nm. Fig. 3 shows reflection spectra of ZnO powder layers with adsorbed RB, EO, FL, CV, and BG dyes. By stacking these five dyes, photocurrent spectra were widened to cover the wavelength range from ~450 to 700 nm. In Fig. 4(a), photocurrent spectra measured following each growth step are shown for a light-harvesting antenna of [ZnO/EO/CV/RB]. When EO was deposited on a ZnO surface during Step 1, large photocurrents were generated over the wavelength region from ~500 to ~550 nm. When CV was deposited on EO in Step 2, photocurrents caused by CV were generated in the wavelength region longer than ~550 nm in addition to the photocurrent from EO absorption. When RB was deposited on CV during Step 3, photocurrents caused by RB were observed around ~570 nm. These results suggest that the multi-dye stacked light-harvesting antenna is effective to widen the wavelength region for sensitization of ZnO. It is important to arrange the dye molecules so that an energy-level scheme with a gradual energy slope similar to that of light-harvesting antennas in plants is formed. This facilitates electron transfer from the multi-dye stacked structure to the semiconductor to achieve high-efficiency sensitization. Fig. 4(b) shows an energy-level scheme determined from photoemission yield spectroscopy in air (PYSA)6measurements for [ZnO/EO/CV/RB]. The direction of the energy slope is opposite to the preferred direction. As a future challenge, to further improve sensitization efficiency, the energy-level scheme of the dye molecules should be optimized. References 1 B. O’Regan and M. Gratzel, Nature 353, 737 (1991). 2 T. Yoshimura, H. Watanabe, and C. Yoshino, J. Electrochem. Soc., 58, 51 (2011). 3T. Yoshimura, Japanese Patent, Tokukai Hei3-60487 (1991) [in Japanese]. 4T. Yoshimura, Japanese Patent, Tokukai 2009-60487 (2009) [in Japanese]. 5 H. Meier, J. Phys. Chem. 69, 719 (1965). 6 Y. Nakajima, D. Yamashita, A. Ishizaki, B. Pellissier, and M. Uda, Mater.Res. Symp. Proc. 1029, 1029-F04-02 (2008). Figure 1

Nanometric molecular separation measurements by single molecule photobleaching

Although considerable progress has been made in imaging distances in cells below the diffraction limit using FRET and super-resolution microscopy, methods for determining the separation of macromolecules in the 10–50nm range have been elusive. We have developed fluorophore localisation imaging with photobleaching (FLImP), based on the quantised bleaching of individual protein-bound dye molecules, to quantitate the molecular separations in oligomers and nanoscale clusters. We demonstrate the benefits of using our method in studying the nanometric organisation of the epidermal growth factor receptor in cells.

Spectroscopic study of ordered hybrid complexes formation between dye aggregates and ReVO4:Eu3 + (Re = Y, Gd, La) nanoparticles

For the first time we report on ordered nano-sized hybrid complexes formation between fluorescent aggregates of potential-sensitive mitochondria probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) and ReVO4:Eu3+ nanoparticles (NPs) in aqueous solutions. To study the efficiency and driving forces of the dye aggregates/NPs complex formation, we analyzed the effects of the dye molecular structure on its aggregation behavior using JC-1 derivatives with different alkyl chain lengths and functional groups (JC-1/C4, JC-1/C3S). The aggregation behavior of the dyes in the solutions either with or without NPs has been analyzed within the Kasha exciton model framework. It was found that depending on the dye structure herringbone-type aggregation (for JC-1) and J-type aggregation (for JC-1/C4 dye) can be observed. The interactions with ReVO4:Eu3+ NPs take place due to Coulomb forces and provoke changes of the aggregate parameters such as the angle between polarization axes of the unit molecules and the line of molecular centers (θ), angle between transition dipole moments of the individual dye molecules (α) and center-to-center distance between molecules (R). The effect of NP shapes (spherical, spindle-like and rod-like) on the structure of hybrid complexes is studied. For the first time, using a microspectroscopic technique we demonstrate the JC-1 aggregates/NPs complex accumulation within the hepatocyte nucleus region in vitro experiment rather than a typical JC-1 specific accumulation in cell mitochondria.

Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes

Several approaches have been described to fluorescently label and image DNA and chromatin in situ on the single-molecule level. These superresolution microscopy techniques are based on detecting optically isolated, fluorescently tagged anti-histone antibodies, fluorescently labeled DNA precursor analogs, or fluorescent dyes bound to DNA. Presently they suffer from various drawbacks such as low labeling efficiency or interference with DNA structure. In this report, we demonstrate that DNA minor groove binding dyes, such as Hoechst 33258, Hoechst 33342, and DAPI, can be effectively employed in single molecule localization microscopy (SMLM) with high optical and structural resolution. Upon illumination with low intensity 405 nm light, a small subpopulation of these molecules stochastically undergoes photoconversion from the original blue-emitting form to a green-emitting form. Using a 491 nm laser excitation, fluorescence of these green-emitting, optically isolated molecules was registered until “bleached”. This procedure facilitated substantially the optical isolation and localization of large numbers of individual dye molecules bound to DNA in situ, in nuclei of fixed mammalian cells, or in mitotic chromosomes, and enabled the reconstruction of high-quality DNA density maps. We anticipate that this approach will provide new insights into DNA replication, DNA repair, gene transcription, and other nuclear processes.

Single‐Molecule Metal‐Induced Energy Transfer (smMIET): Resolving Nanometer Distances at the Single‐Molecule Level

We present a new concept for measuring distance values of single molecules from a surface with nanometer accuracy using the energy transfer from the excited molecule to surface plasmons of a metal film. We measure the fluorescence lifetime of individual dye molecules deposited on a dielectric spacer as a function of a spacer thickness. By using our theoretical model, we convert the lifetime values into the axial distance of individual molecules. Similar to Förster resonance energy transfer (FRET), this allows emitters to be localized with nanometer accuracy, but in contrast to FRET the distance range at which efficient energy transfer takes place is an order of magnitude larger. Our technique can be potentially used as a tool for measuring intramolecular distances of biomolecules and complexes.

Impact of local compressive stress on the optical transitions of single organic dye molecules

The ability to mechanically control the optical properties of individual molecules is a grand challenge in nanoscience and could enable the manipulation of chemical reactivity at the single-molecule level. In the past, light has been used to alter the emission wavelength of individual molecules or modulate the energy transfer quantum yield between them. Furthermore, tensile stress has been applied to study the force dependence of protein folding/unfolding and of the chemistry and photochemistry of single molecules, although in these mechanical experiments the strength of the weakest bond limits the amount of applicable force. Here, we show that compressive stress modifies the photophysical properties of individual dye molecules. We use an atomic force microscope tip to prod individual molecules adsorbed on a surface and follow the effect of the applied force on the electronic states of the molecule by fluorescence spectroscopy. Applying a localized compressive force on an isolated molecule induces a stress that is redistributed throughout the structure. Accordingly, we observe reversible spectral shifts and even shifts that persist after retracting the microscope tip, which we attribute to transitions to metastable states. Using quantum-mechanical calculations, we show that these photophysical changes can be associated with transitions among the different possible conformers of the adsorbed molecule.

Feedback-controlled electro-kinetic traps for single-molecule spectroscopy

A principal limitation of single-molecule spectroscopy in solution is the diffusion-limited residence time of a given molecule within the detection volume. A common solution to this problem is to immobilize molecules of interest on a passivated glass surface for extending the observation time to obtain reliable data statistics. However, surface tethering of molecules often introduces artifacts, particularly when studying the structural dynamics of biomolecules. To circumvent this limitation, we investigated alternative ways to extend single-molecule observation times in solution without surface immobilization. Among various possibilities, the so-called anti-Brownian electro-kinetic trap (or ABEL trap) seems best suited to achieve this goal. The essential part of that trap is a feedback-controlled electro-kinetic steering of a molecule’s position in reaction to its diffusive Brownian motion which is monitored by fluorescence, thus keeping the molecule within a sub-micron sized detection volume. Fluorescence trace recordings of over thousands of milliseconds duration on individual dye molecules within an ABEL trap have been reported. In this short review, we shall briefly discuss the principle and some results of ABEL trapping of individual molecules with possible extensions to future works.

Slow Single-Molecule Diffusion in Liquid Crystals

We report on measurements of single-molecule Brownian motion in liquid crystals, unravelling the anisotropic mobility of individual dye molecules. This anisotropic Brownian motion is directly correlated with the structural properties in a smectic A (8CB) and a nematic (5CB) liquid crystal sample cell on the micrometer scale using polarization contrast microscopy. A considerably slower mobility of dye molecules is found as compared to self-diffusion measurements by NMR, while anisotropy values compare well to recent literature data. This is suggested to be related to local distortions of the director structure around the dye molecules.

Dye molecules as single-photon sources and large optical nonlinearities on a chip

We point out that individual organic dye molecules, deposited close to optical waveguides on a photonic chip, can act as single-photon sources. A thin silicon nitride strip waveguide is expected to collect 28% of the photons from a single dibenzoterrylene molecule. These molecules can also provide large, localized optical nonlinearities, which are enough to discriminate between one photon or two through a differential phase shift of 2° per photon. This new atom–photon interface may be used as a resource for processing quantum information.

Quantitative Fluorescence Microscopy To Determine Molecular Occupancy of Phospholipid Vesicles

Encapsulation of molecules in phospholipid vesicles provides unique opportunities to study chemical reactions in small volumes as well as the behavior of individual proteins, enzymes, and ribozymes in a confined region without requiring a tether to immobilize the molecule to a surface. These experiments generally depend on generating a predictable loading of vesicles with small numbers of target molecules and thus raise a significant measurement challenge, namely, to quantify molecular occupancy of vesicles at the single-molecule level. In this work, we describe an imaging experiment to measure the time-dependent fluorescence from individual dye molecules encapsulated in ~130 nm vesicles that are adhered to a glass surface. For determining a fit of the molecular occupancy data to a Poisson model, it is critical to count empty vesicles in the population since these dominate the sample when the mean occupancy is small, λ ≤ ~1. Counting empty vesicles was accomplished by subsequently labeling all the vesicles with a lipophilic dye and reimaging the sample. By counting both the empty vesicles and those containing fluors, and quantifying the number of fluors present, we demonstrate a self-consistent Poisson distribution of molecular occupancy for well-solvated molecules, as well as anomalies due to aggregation of dye, which can arise even at very low solution concentrations. By observation of many vesicles in parallel in an image, this approach provides quantitative information about the distribution of molecular occupancy in a population of vesicles.

An Easy Method To Monitor Lactide Polymerization with a Boron Fluorescent Probe

Fluorescence spectroscopy has been widely used to monitor different polymer processes such as polymerization kinetics, chain entanglements, and thermal transitions. The solvent-free controlled ring-opening polymerization (ROP) of lactide is significant both commercially and for research; thus, monitoring this process with a simple fluorescence method can be very useful. Here, a fluorescent dye, difluoroboron 4-methoxydibenzoylmethane (BF(2)dbmOMe) is employed to probe lactide bulk ROP by measuring the emission from solidified reaction aliquots at room temperature. It was found that, through the course of polymerization, the fluorescence of BF(2)dbmOMe in the solid-state aliquots exhibited a systematic shift from yellow to green and then to blue, accompanied by a gradual reduction in the decay lifetime. The fluorescence color change is sensitive to the monomer percent conversion, not the polymer molecular weight. On the basis of these observations and experimental data, we propose that the long-wavelength emission with perceivably longer lifetimes arises from BF(2)dbmOMe dye aggregates (ground and/or excited states), while the dissolved individual dye molecules are responsible for the blue fluorescence with a shorter lifetime. This demonstration of the utility of BF(2)dbmOMe as a fluorescent probe for lactide polymerization could have important practical implications.